CN108267425A - A kind of method by resonance rayleigh light scattering method Accurate Determining chitosan content - Google Patents

Abstract

The present invention relates to a kind of methods by resonance rayleigh light scattering method Accurate Determining chitosan content, belong to macromolecular substances detection field.In order to which the content for overcoming chitosan in finished product is difficult to quantify, the cumbersome technical deficiency of operating technology, the present invention provides a kind of method by resonance rayleigh light scattering method Accurate Determining chitosan content, this method, under mildly acidic conditions with luring the red characteristic for reacting generation associated matter, measures the RRS intensity Is of ionic associate using chitosan at maximum RRS wavelength_RRSWith the RRS intensity Is of reagent blank_0RRS, calculate Δ I_RRS=I_RRS‑I_0RRS, and then the standard curve of △ I and different molecular weight chitosan concentration are established, then according to the molecular weight selection criteria curve of chitosan in sample, according to the Δ I of the sample measured_RRSThe content of chitosan in sample is calculated.This method has the advantages of reagent is cheap, high sensitivity favorable reproducibility, is suitble to promote the use of in practical applications.

Description

A kind of method by resonance rayleigh light scattering method Accurate Determining chitosan content

Technical field

The present invention relates to a kind of methods by resonance rayleigh light scattering method Accurate Determining chitosan content, belong to macromolecular complex Quality detection field.

Background technology

Chitosan (chitosIn) is the catabolite of chitin.Chemical name for Chitosan (1,4) -2- amino - 2- deoxidation-D- glucans, be shell-fish (shrimp, crab) animal, insect ectoskeleton main component, chitosan is in nature point Cloth is extensive, belongs to macromolecule green material.In relation to the mankind to the understanding of chitosan since 19th century, to 60 years this century In generation, just becomes to become increasingly active to the research of chitin/chitosan.In the world, since 1894,6 related first had been held The meeting of shell element/chitosan；First chitin academic conference was also held in Asia in 1994, had been held 2 times.And China is only It is that just the exploitation of chitin/chitosan and application are paid attention in recent years.In October, 1996 has held first in Dalian Chitin chemistry academic conference.Chitosan and its derivative has wide answer in industries such as food, medicine, environmental protection, chemical industry Use prospect.Chitosan has good biocompatibility and bioactivity, nontoxic, harmless, without challeng, has to human body Reinforced immunological inhibits aging, prevents disease, promote disease recovery from illness, adjust physiological function five functional.Chitosan production on the market Product are irregular, and formula differs, and the content shared by chitosan directly influences the performance of its function.Therefore, it is accurately fixed to establish Analysis method, it is all most important to the stability and controllability that ensure product quality.

At present, chitosan analysis and research and method for measuring mainly have spectrophotometry, fluorescence analysis, electrochemical process, Infra-red sepectrometry survey, liquid chromatography, gas chromatography etc..As Fluorimetric Quenching Method for Determination chitosan is established in S. Korea and the USA's Na et al. research The content of chitosan in gel mould has significantly fluorescein isothiocynate (FITC) in PBS buffer solutions using chitosan Fluorescent quenching effect, and fluorescent quenching degree and the amount of chitosan are in a linear relationship in a certain range, glimmering so as to establish Optical quenching method measures chitosan content.Quotient army and two people of Wang Bei are established in high effective liquid chromatography for measuring raw material and compound premix Oligopolymerization chitosan sugared content.Tan Xuecai, Mai Zhibin et al. establish with alizarin red (IR) for electroactive probe indirect determination without electricity The new method of the chitosan (chitosan) of activity.Since chitosan itself is easily degraded, and UV absorption is weaker, is often adopted in experiment Content analysis is carried out with sour water solution indirect colourimetry, the concentrated sulfuric acid is usually used in the method, operation has certain danger, and And be not suitable for the analysis detection of micro chitosan content.It is right before the assay that gas chromatography generally requires when measuring chitosan content Chitosan performs the derivatization processing, and operation is complex.It is generally also first to gather shell when HPLC methods measure for chitosan content Sugar is measured again after carrying out sour water solution.These sour water solutions or derivatization operating process all can to the measurement result of chitosan content There is larger impact, some methods for directly measuring chitosan content gradually attract attention.

Spectrophotometry, Fluorimetric Quenching Method and the electrochemical process sensitivity that approach described above has been reported at present are all inadequate It is high.And then there is the shortcomings of time-consuming, the cost is relatively high in liquid chromatography, nuclear magnetic resonance, mass spectrography etc..It is and domestic at present Outside, the correlative study for directly measuring chitosan content using easy, quick, high sensitivity Resonance Rayleigh Scattering Technique is less, And it has many advantages, such as high sensitivity, simplicity, quick and can measure at normal temperatures and pressures, it can be in common fluophotometer Upper realize measures, and the probe species available for measure are various, and therefore, resonance rayleigh light scattering method, which measures chitosan, very big grind Study carefully space and significance.

Resonance rayleigh light scattering method (_RRS) it is the new analytical technology to grow up in recent years, as a kind of new analytical technology It is more and more studied and is applied, it is also used for shell other than it can be used for trace metal, nonmetallic and organic matter measure The research of glycan.With_RRSIn the method for measuring chitosan, mainly contaminated using dyestuff such as azogeramine, xylenol orange, reactive orange Material, orchid receive between free and easy sauce red B (IBB), nylon mountain Huang N-3RL (NYN), orange beta-naphthol, reactive brilliant bule, acid violet existing be total to It shakes scattering.And chitosan is with luring quantitative analysis of the associated matter that red combination is formed for chitosan using there is not been reported.

Invention content

In the prior art the content of chitosan in finished product is difficult to quantify to overcome, the cumbersome technology of operating technology is not Foot, the present invention provide a kind of method by resonance rayleigh light scattering method Accurate Determining chitosan content, and the present invention utilizes chitosan Under mildly acidic conditions with luring the red RRS intensity reacted generation associated matter, ionic associate is measured at maximum RRS wavelength I_RRSWith the RRS intensity Is of reagent blank_0RRS, calculate Δ I_RRS=I_RRS-I_0RRS, establish △ I and different molecular weight chitosan concentration Then standard curve selects suitable standard curve, according to the Δ of the sample measured according to the molecular weight of chitosan in sample I_RRS, influenced so as to which content this method of chitosan in sample measure be calculated by the molecular weight of chitosan, with reagent Inexpensively, the advantages of high sensitivity favorable reproducibility, it is suitble to promote the use of in practical applications.

A kind of method by resonance rayleigh light scattering method Accurate Determining chitosan content includes the following steps：

1) standard curve of the chitosan concentration of △ I and different molecular weight is drawn:B-R bufferings are added in into 10ml colorimetric cylinders Liquid 1.00ml, the low molecular chitosan standard solution or appropriate sample liquid of a certain concentration gradient, polysorbas20 solution 0.80ml are lured Puzzled red solution 1.00ml, B-R buffer solution 1.50ml are diluted with water to scale and shake up, on sepectrophotofluorometer, with λ Ex=λ em synchronize scanning, record Resonance Rayleigh Scattering Spectra, then ionic associate is measured at maximum RRS wavelength RRS intensity Is_RRSWith the RRS intensity Is of reagent blank_0RRS, calculate Δ I_RRS=I_RRS-I_0RRS, establish △ I and low molecular chitosan The standard curve C1 of concentration；Low molecular chitosan solution is replaced using middle-molecular-weihydroxyethyl chitosan solution, establishes △ I and middle molecule shell The standard curve C2 of Glycan concentration；Low molecular chitosan solution is replaced using high molecular weight chitosan solution, establishes △ I and high score The standard curve C3 of seed chitosan concentration；

Under best experiment condition, measurement result of the system to the chitosan of different molecular weight is investigated.It investigates respectively The chitosan of low molecular weight, middle-molecular-weihydroxyethyl and high molecular weight.By statistical analysis, the chitosan result of different molecular weight is deposited In the significance difference opposite sex, this method measures chitosan content to be influenced by different molecular weight.

2) preparation of the sample working solution of 10 μ g/mL：The capsule shells of a certain amount of detected sample are weighed, it is molten using glacial acetic acid It solves and its constant volume is obtained into stock sample solution, filter storing solution with funnel absorbent cotton, filtrate is through 6000r/min, centrifuge 20min takes supernatant 2.5mL in 100mL volumetric flasks, constant volume, obtains the sample working solution of a concentration of 10 μ g/mL.

3) according to chitosan molecule amount selection criteria curve：△ I are drawn using step 1) kind detection method and sample works The standard curve of liquid, and by it compared with the chitosan standard curve of different molecular weight, according to the △ I of sample and sample working solution Standard curve regression equation determine chitosan in sample molecular weight size, according to the molecule of corresponding chitosan The size of amount determines used regression equation；

4) chitosan content determines in sample：Sample working solution 1mL is taken, is measured by step 1) experimental method, Its absorbance value I is measured at 343nm, and calculates Δ I_RRS=I_RRS-I_0RRS, △ I substitution equations of linear regression are acquired in sample The content of chitosan, while do mark-on reclaims experiment.

As described above by the method for resonance rayleigh light scattering method Accurate Determining chitosan content, the B-R buffer solutions PH for 5.0, applicants experimentally found that, for system in the B-R buffer solution medium sensitivity highests of pH=5.0, Δ I values are maximum, In addition it is found by the applicant that glycine-HCI has preferable Anti-Jamming, energy masked portion metal ion, therefore optimized buffer is molten The B-R buffer solutions of liquid selected as pH=5.0.

As described above by the method for resonance rayleigh light scattering method Accurate Determining chitosan content, solution adds in step 1 Enter sequence to be firstly added chitosan standard solution, secondly add in glycine-HCI buffer solution, add in lure red solution again.

As described above by the method for resonance rayleigh light scattering method Accurate Determining chitosan content, it is cooled to room temperature to measure Time be 10min-2h.

As described above by the method for resonance rayleigh light scattering method Accurate Determining chitosan content, the concentration range of chitosan The μ g/mL of 0.5 μ g/mL~4.5

Sample pre-treatments：Capsule shells to be detected are taken, weigh a certain amount of capsule shells, sample is obtained using glacial acetic acid dissolving constant volume Storing solution filters storing solution with funnel absorbent cotton, filtrate through 6000r/min, centrifuge 20min, take supernatant 2.5mL in 100mL volumetric flasks, constant volume obtain the sample working solution of a concentration of 10 μ g/mL；

With sample working solution, empirically method draws sample curves, compared with the chitosan standard curve of different molecular weight, The size of the molecular weight of the chitosan in sample is determined according to the regression equation of sample, according to the molecular weight of corresponding chitosan Size determine used in △ I and chitosan concentration standard curve；

Sample working solution 1mL is taken, empirically method is measured, its absorbance value I is measured at 343nm, and calculate Δ I_RRS=I_RRS-I0_RRS, △ I substitution equations of linear regression are acquired into the content of chitosan in sample, while do mark-on reclaims experiment.

Test example：

1. the RRS spectrum of the red-CTS associated matters of temptation

Respectively to the red, red-CTS associated matters of temptation is lured to be carried out with λ ex=λ em on F-2500 sepectrophotofluorometers Synchronous scanning obtains RRS spectrum.Red and CTS RRS characteristic spectrums is lured to see Fig. 1.As shown in Figure 1：Lure red reagent blank RRS is extremely weak, CTS with lure red reacting forming ion associated matter after, solution RRS in the range of 250~650nm is caused to enhance, At 343nm, and in a certain range, RRS increases with the increase of CTS contents at maximum RRS peaks.

2. influence of the different buffer solutions for system absorbance

This development test tetra- kinds of citric acid-sodium citrate, B-R buffer solutions, glycine-HCI, HIc-NIIc bufferings are molten Liquid is to system_RRSInfluence.The result shows that temptation is red to react the Δ I in B-R buffer solutions with CTS_RRSIt is larger.Therefore it is believed that B-R buffer systems are most beneficial for the progress of experiment.

Two groups of colorimetric cylinders (standard pipe and reagent blank pipe) are taken, are separately added into the buffer solution B-R buffer solutions of different pH value (1.00ml), then CTS Standard Applying Solutions (10.00 μ g/ml) 3.00ml is added in standard pipe successively, it is red using liquid to add in temptation (2.0 × 10-4mol/L) 1.00ml, reagent blank are not added with CTS, add in the red application liquid (1.0 × 10-4mol/L) of temptation 1.00ml using reagent blank as reference, measures the RRS values of corresponding pH value.Draw Δ I-pH curves, influence table of the pH value to RRS Bright, optimal pH range is 4.50-5.50, tests final selected reaction pH=5.00 (see Fig. 2)

3. influence of the addition of buffer solution to absorbance value

Two groups of colorimetric cylinders (standard pipe and reagent blank pipe) are taken, are separately added into the different amounts of B-R solution of pH=5.00, are marked Quasi- pipe adds in CTS Standard Applying Solutions (10.00 μ g/mL) 3.00ml, lures red application liquid (2.0 × 10-4mol/L) 1.00ml, examination Agent blank is not added with CTS, using reagent blank as reference, measures the RRS of corresponding amount of buffer.Draw Δ I-V curve, the results showed that, With the increase of buffer solution addition, for Δ I in 1.50ml and 4.00ml additions, the changing value of Δ I is little, therefore optimized buffer The dosage of liquid chooses 1.50mL (see Fig. 3).

4. the influence of the red addition of temptation

Two groups of colorimetric cylinders (standard pipe and reagent blank pipe) are taken, standard pipe adds in the B-R buffer solution 1.50ml of pH=5.00, CTS (10.00 μ g/ml) 3.00ml, reagent blank be not added with luring it is red, then be separately added into it is different amounts of temptation it is red, with reagent blank For reference, the RRS for accordingly luring red concentration is measured.Draw Δ I-V curve, the results showed that (see Fig. 4) is lured in 10ml colorimetric cylinders When the volume of red (2.0 × 10-4mol/L) is 1.00ml, that is, a concentration of 2 × 10-5mol/L when Δ I_RRSIntensity reaches maximum, uses It measures excessive or very few can lead to Δ I_RRSValue reduces.Obviously, lure red dosage very few, reaction is incomplete；Dosage excessively also can shadow CTS is rung with luring red ion association, so as to cause RRS strength reductions.(see Fig. 4)

5. influence of the reaction temperature for system absorbance

2 groups of 10ml colorimetric cylinders (standard pipe and reagent blank pipe) are taken, standard pipe adds in pH=5.00B-R buffer solutions 1.50ml lures red (2.0 × 10-4mol/L) 1.00ml, CTS (10.00 μ g/mL) 3.00ml, and blank tube is not added with CTS, simultaneously Water-bath 3min.Measuring bath temperature is：20,40,60,80,100 DEG C of room temperature, the Δ I of system.It is mapped with Δ I-Tem.As a result table Bright (see Fig. 5), temperature is affected to the reaction, under certain condition, the reaction effect in a certain range of (80 DEG C) below room temperature Fruit is best, and with the raising of temperature, Δ I rises, and obtains high value at 80 DEG C.Experiment control should be in 80 DEG C of fire-bars It is carried out under part.

6. influence of the heating time for system absorbance

Under experimental conditions, influence of the heating time to system has been investigated, has as a result seen Fig. 7.Consider heating time long appearance Intermolecular association is easily influenced, so it is 3min, 5min, 10min to select reaction heating time, the results show that in three heating The Δ I differences of time are little, but under equal conditions, the Δ I of 5min heating times is compared with other two groups of highers.

7. influence of the sensitizer for system absorbance

This development test dodecyl sodium sulfate (SLS), lauryl sodium sulfate (SDS), polysorbas20 (Tween20), Tween 80 (Tween 80), polyvinyl alcohol, influences of the OP-10 to system RRS, as shown in Figure 6.Wherein SLS's probes into experiment, It was found that the concentration that the reason of occurring cotton-shaped jelly in reagent, this situation occur may be sensitizer SLS larger causes.As a result table It is bright, temptation it is red with CTS to react the effect of enhanced sensitivity in polysorbas20 (Tween 20) solution best, therefore select behind polysorbas20 continuation Experiment.

8. the influence of polysorbas20 dosage

Two groups of colorimetric cylinders (standard pipe and blank tube) are taken, are separately added into pH=5.00B-R buffer solutions (1.50ml), then according to It is secondary to add in CTS Standard Applying Solutions (10.00 μ g/mL) 3.00ml in standard pipe, add in the red application liquid (2.0 × 10 of temptation^-4mol/L) 1.00ml and different amounts of polysorbas20 solution, blank tube are not added with sensitizer, add in pH=5.00 B-R buffer solutions (1.50ml), then CTS Standard Applying Solutions (10.00 μ g/ml) 1.00ml is added in standard pipe successively, it is red using liquid to add in temptation (2.0×10^-4Mol/L) 1.00ml using this blank tube as reference, measures the RRS values of polysorbas20 respective volume.It is bent to draw Δ I-V Line, influence of the polysorbas20 dosage to RRS shows the dosage of best polysorbas20 when being 0.8ml, i.e. during 800 μ g/ml, effect compared with It is good.

9. influence of the reagent addition sequence for system absorbance

It tests 12 kinds and adds in order (fully shaken up after often adding in a kind of solution, then add another solution)：It is shown in Table 1. Addition is：The B-R buffer solutions 1.50ml of pH=5.00.CTS Standard Applying Solutions (10.00 μ g/mL) 1.00mL, lures red application Liquid (2.0 × 10^-4Mol/L) 1.00ml, the experimental results showed that the Δ I of " CTS standards+polysorbas20+temptation red+buffer solution "_RRSMost Greatly, this kind addition order effect is optimal, and experimental result is as shown in Figure 8.

The influence (10.00 μ g/ml of CTS) of 1 reagent addition sequence of table

10. influence of the stabilization time for system absorbance

Take 4 groups of 50mL volumetric flasks (standard group and reagent blank pipe), stable experiment under the conditions of being protected from light and not being protected from light respectively Time probes into, and concrete operations are spat for pH=5.00 standard pipes is taken to add in CTS Standard Applying Solutions (10.00 μ g/mL) 15.00ml Warm 204.00ml lures red application liquid (2.0 × 10^-4Mol/L) 5.00ml, B-R buffer solution 7.50ml, blank tube are not added with CTS, survey Surely the Δ I of system after 5,10,20,30,40,60,80,120min is placed.It is mapped with Δ I-t, the results showed that (see Figure 10), reaction It is not protected from light and is keeing relative stability in 2h.Therefore experimental selection is not protected from light condition, measures and finishes in 2h.

11. influence of the ionic strength for system absorbance

In experiment, the influence to the RRS values of the reaction has been investigated with NaCl (0~0.3mol/L).Take 2 groups of 10ml colorimetric cylinders (experiment tube and control tube) is sequentially separately added into CTS titers (10.00 μ g/mL) 2.00ml, polysorbas20 0.80ml, Buffer solution B-R buffer solution the 1.50ml of red (2.0 × 10-4mol/L) 1.00ml, pH=5.00 are lured, experiment tube adds in certain dense The NaCl of degree, blank tube are not added with NaCl.The result shows that (such as Figure 11), △ I are after first subtracting with the general trend that NaCl concentration changes Slow to rise again, in 0-0.05mol/L, interior react is tended to be steady in negatively influencing, and possible cause is that NaCl and CTS is competed so that Δ I_RRSDecline.

The present invention has following technical advantage compared with prior art：

1) good linearity, detection limit are low：Under optimum experimental condition, according to the chitosan pair of determination of experimental method various concentration The △ I answered draw standard curve, the results showed that, there are good with △ I in the range of the μ g/mL of 0.5 μ g/mL of concentration~4.5 for chitosan Good linear relationship, 0.4987 μ g/mL of detection limit.

2) this method measure is influenced by the molecular weight of chitosan, in actual sample measure, need to be considered as close point The standard items of son amount do quantitative criterion, have the advantages of reagent is cheap, high sensitivity favorable reproducibility.

Description of the drawings

Fig. 1 is the abosrption spectrogram under different CTS concentration that CTS- lures corpus hemorrhagicum system.

Wherein pH=3.50B-R buffer solutions+2.0 × 10^-4Mol/L lures red solution, is denoted as a；1---I+0.50μg/mL Lure red solution；2---I+1.0 μ g/mL lure red solution；3---I+1.5 μ g/mL lure red solution；4---I+2.0 μ g/mL are lured Puzzled red solution；5---I+3.0 μ g/mL lure red solution.

Influence figure of Fig. 2 differences buffer solution for system absorbance.

Fig. 3 is influence of the addition of B-R buffer solutions to Δ I.

Fig. 4 is to lure influence of the red solution addition to Δ I.

Fig. 5 is influence of the reaction temperature for Δ I.

Fig. 6 is influence of the sensitizer for Δ I.

Influence of Fig. 7 reaction temperatures for Δ I.

Influence of Fig. 8 reagents addition sequence for Δ I.

Influence of Fig. 9 heating times for Δ I

Influence of Figure 10 stabilization times for Δ I.

Influence of Figure 11 ionic strengths for Δ I.

Specific embodiment

The present invention is further described, but the invention does not limit the present invention in any way below by way of specific embodiment The range of patent protection.

Embodiment

A kind of method by resonance rayleigh light scattering method Accurate Determining chitosan content includes the following steps：

1) standard curve of the chitosan concentration of △ I and different molecular weight is drawn:

B-R buffer solution 1.00ml, the low molecular chitosan standard solution of a certain concentration gradient are added in into 10ml colorimetric cylinders Or appropriate sample liquid, polysorbas20 solution 0.80ml, red solution 1.00ml, B-R buffer solution 1.50ml is lured to be diluted with water to Scale simultaneously shakes up, and on sepectrophotofluorometer, scanning is synchronized with λ ex=λ em, records Resonance Rayleigh Scattering Spectra, then The RRS intensity Is of ionic associate are measured at maximum RRS wavelength_RRSWith the RRS intensity Is of reagent blank_0RRS, calculate Δ I_RRS=I_RRS-I_0RRS, establish the standard curve C1 of △ I and low molecular chitosan concentration；It is replaced using middle-molecular-weihydroxyethyl chitosan solution Low molecular chitosan solution is changed, establishes the standard curve C2 of △ I and middle molecular chitosan concentration；It is molten using high molecular weight chitosan Liquid replaces low molecular chitosan solution, establishes the standard curve C3 of △ I and polymer chitosan concentration；

Under best experiment condition, measurement result difference of the system to the chitosan of different molecular weight is investigated.Respectively The chitosan of low molecular weight, middle-molecular-weihydroxyethyl and high molecular weight has been investigated, the results are shown in Table 2.By statistical analysis, different molecular weight Chitosan result there are significance difference the opposite sex, this method measure chitosan content influenced by different molecular weight.

The chitosan result of 2 different molecular weight of table

2) preparation of the sample working solution of 10 μ g/mL：Australia profit dimension crust cellulose capsule is removed photoresist softgel shell, weigh 0.04g in It in 100mL volumetric flasks, is dissolved with 0.5mol/L glacial acetic acid, constant volume obtains stock sample solution.Storing solution, filter are filtered with funnel absorbent cotton Liquid is through 6000r/min, centrifuge 20min, and taking supernatant 2.5mL, constant volume obtains a concentration of 10 μ g/mL in 100mL volumetric flasks Sample working solution.

3) according to chitosan molecule amount selection criteria curve：△ I are drawn using step 1) kind detection method and sample works The standard curve of liquid, and by it compared with the chitosan standard curve of different molecular weight, according to the △ I of sample and sample working solution Standard curve regression equation determine chitosan in sample molecular weight size, according to the molecule of corresponding chitosan The size of amount determines used regression equation；

With sample working solution, empirically method draws sample curves, compared with the chitosan standard curve of different molecular weight, As a result be △ I=0.0459c+0.0097 for the equation of linear regression of sample, related coefficient 0.999, as a result with low molecular weight Chitosan standard curve approaches, using the standard curve of low-molecular weight chitoglycan as quantitation curves.

4) chitosan content determines in sample：With sample working solution, empirically method draws sample curves, with different molecular The chitosan standard curve of amount compares, and result is Δ I=232.18c+69.502 for the equation of linear regression of sample, related coefficient 0.9947, it is as a result approached with the chitosan standard curve of high molecular weight, using the standard curve of middle-molecular-weihydroxyethyl chitosan as fixed Measure standard curve.

Sample working solution 1.00mL is taken, empirically method is measured, its scattering value I is measured at 348nm, and calculate △ I=I-I₀, △ I substitution equations of linear regression are acquired into the content of chitosan in sample, while do mark-on reclaims experiment.As a result For：The content of chitosan is 871.16mg/g, RSD 3.40% in Australia's profit dimension crust cellulose capsule.Recovery of standard addition is shown in Table 3.

3 sample analysis result of table

With optimal conditions, CTS standards (10.00 μ g/ml) 3.00ml, polysorbas20 0.80ml, temptation are red successively for experiment (1.0×10^-4Mol/L) 1.00ml, B-R buffer solution 1.50ml form stable ionic associate, 80 DEG C of heating water bath 5min. After 5min, cooling can start with F-2500 type sepectrophotofluorometers with λ_ex=λ_emSweep measuring is synchronized, is measured in 2h It completes.There are good linear relationships with △ I in the range of 0.5 μ g/ml-4 μ g/ml of concentration for chitosan, and the range of linearity is wide, detection It is limited to 0.180 μ g/ml.This method measure is influenced by the molecular weight of chitosan, in actual sample measure, need to be considered as phase The standard items of nearly molecular weight do quantitative criterion, have that reagent is cheap, high sensitivity, favorable reproducibility and it is easy to operate the advantages that.

3.3 ranges of linearity and detection limit

Linear relationship and detection limit (DL)

In a series of 10ml colorimetric cylinders, be sequentially separately added into CTS titers (10.00 μ g/ml) (0.50, 1.00th, 2.00,3.00,4.00,5.00,5.50ml), polysorbas20 0.80ml, temptation red (2.0 × 10^-4Mol/L) 1.00ml, pH =5.00 buffer solution B-R buffer solution 1.50ml, are diluted to graduation mark with distilled water, shake up.Being prepared into concentration gradient is The standard solution of (0.50,1.00,2.00,3.00,4.00,5.00,5.50 μ g/ml), while reference is made with reagent blank, it surveys RRS makees linear trend figure with Δ I-c.The result shows that in the range of 0.50~4.00 μ g/ml, RRS intensity Δ I and CTS concentration cs In good linear relationship, linear equation is in B-R buffer systems：Δ I=210.8c+43.6, R²=0.9916.

Selection blank tube, minimum concentration pipe (0.50 μ g/ml) do 13 respectively.According to formula：

DL=3S_△I/L

All substitute into obtain DL=0.180 μ g/ml

It is above-mentioned statistics indicate that the method high sensitivity, the advantages of detection limit is low, be that Resonance scattering measures the good of chitosan Good supplement.

The influence of coexisting substances

Empirically method investigated coexistent metallic ion, 10.00 μ g/ml CTS measurement results of inorganic acid radical ion pair shadow It rings, the concentration results for calculating each coexisting substances of generation about ± 5% relative error addition are listed in table 4

The influence (10.00 μ g/mL of CTS) of 4 coexisting ion of table

As seen from table, coexisting substances such as dextrin, glucose, citric acid, amino acid etc. interferes measurement result smaller.

Claims (7)

1. a kind of method by resonance rayleigh light scattering method Accurate Determining chitosan content includes the following steps：

1) standard curve of the chitosan concentration of △ I and different molecular weight is drawn：B-R buffer solutions are added in into 10ml colorimetric cylinders 1.00ml, the low molecular chitosan standard solution or appropriate sample liquid of a certain concentration gradient, polysorbas20 solution 0.80ml, temptation Red solution 1.00ml, B-R buffer solution 1.50ml, are diluted with water to scale and shake up, on sepectrophotofluorometer, with λ ex =λ em synchronize scanning, record Resonance Rayleigh Scattering Spectra, then ionic associate is measured at maximum RRS wavelength RRS intensity Is_RRSWith the RRS intensity Is of reagent blank_0RRS, calculate Δ I_RRS=I_RRS-I_0RRS, it is dense with low molecular chitosan to establish △ I The standard curve C1 of degree；Low molecular chitosan solution is replaced using middle-molecular-weihydroxyethyl chitosan solution, △ I is established and gathers with middle molecule shell The standard curve C2 of sugared concentration；Low molecular chitosan solution is replaced using high molecular weight chitosan solution, establishes △ I and macromolecule The standard curve C3 of chitosan concentration；

2) preparation of the sample working solution of 10 μ g/mL：The capsule shells of a certain amount of detected sample are weighed, using glacial acetic acid dissolving simultaneously Its constant volume is obtained into stock sample solution, filters storing solution with funnel absorbent cotton, filtrate is through 6000r/min, centrifuge 20min takes supernatant 2.5mL in 100mL volumetric flasks, constant volume, obtains the sample working solution of a concentration of 10 μ g/mL；

3) according to chitosan molecule amount selection criteria curve：△ I and sample working solution are drawn using detection method in step 1) Standard curve, and by it compared with the chitosan standard curve of different molecular weight, according to the △ I of sample and the mark of sample working solution The regression equation of directrix curve determines the size of the molecular weight of the chitosan in sample, according to the molecular weight of corresponding chitosan Size determines used regression equation；

4) chitosan content determines in sample：Sample working solution 1mL is taken, is measured by step 1) experimental method, in maximum RRS Its absorbance value I is measured at wavelength, and calculates Δ I_RRS=I_RRS-I_0RRS, △ I substitution equations of linear regression are acquired into shell in sample The content of glycan, while do mark-on reclaims experiment.

2. the method according to claim 1 by resonance rayleigh light scattering method Accurate Determining chitosan content, feature exists In the maximum RRS wavelength is 343nm.

3. the method according to claim 1 by resonance rayleigh light scattering method Accurate Determining chitosan content, feature exists In the pH of the B-R buffer solutions is 5.0.

4. the method according to claim 1 by resonance rayleigh light scattering method Accurate Determining chitosan content, feature exists --- polysorbas20 --- temptation is red --- buffering in the addition sequence of each solution is chitosan standard solution in the step 1) Liquid.

5. the method according to claim 1 by resonance rayleigh light scattering method Accurate Determining chitosan content, feature exists In, after being diluted with water to scale and shaking up by system be placed in 80 DEG C of water-baths heat 5min be subsequently cooled to room temperature.

6. the method according to claim 5 by resonance rayleigh light scattering method Accurate Determining chitosan content, feature exists In it is 10min-2h to be cooled to room temperature to the time of measure.

7. the method according to claim 1 by resonance rayleigh light scattering method Accurate Determining chitosan content, feature exists In the μ g/mL of 0.5 μ g/mL of concentration range~4.5 of, chitosan.